A long-term goal of the Leavitt laboratory is to understand the molecular basis of megakaryocytopoiesis, the developmental process whereby pleuripotent hematopoietic progenitor cells generate megakaryocyte lineage-committed cells, megakaryocytes, and ultimately circulating platelets. Platelets are critical for maintaining hemostatic integrity, and platelet transfusions provide life-sustaining support for an ever increasing number of thrombocytopenic patients, especially those undergoing chemotherapy and bone marrow transplantation. The growing national problem of platelet shortages highlights the urgent need for novel methods to treat and prevent thrombocytopenia. An improved understanding of megakaryocytopoiesis should help in developing such novel therapies. We are currently funded to study the role of thrombopoietin-induced intracellular signaling in megakaryocytopoiesis using murine model systems (P50-HL54476, Project #2). As part of that work, we recently reported a robust system for generating large numbers of megakaryocytes from rnurine embryonic stem (ES) cells in vitro. We have applied the system to the study of megakaryocytopoiesis and to the study of megakaryocyte integrin signaling. Moreover, the murine system allows one to express genes of interest using retroviral vectors and to suppress gene [unreadable] expression using RNA interference, further demonstrating its utility. A similar system using human ES cells would allow for the direct study of human megakaryocytopoiesis, and should be applicable to other aspects of hematopoiesis. In keeping with the stated purpose of this competitive supplement RFA (PA# PAR-04-37), we are requesting funds to acquire the skills needed to growing human ES cells and to apply those skills to establishing an in vitro system to generate megakaryocytes from human ES cells. We will use the NIH-approved human ES cells lines UC06 (HSF-6) and WA09 (H9). [unreadable] [unreadable] [unreadable]